Microscope for reflected-light and transmitted-light microscopy

ABSTRACT

A microscope is disclosed in which a specimen is arranged between two objectives and can be observed with reflected light as well as with transmitted light. In a microscope with field transmission, two objectives have substantially identical optical characteristics and at least one of the two objectives is followed by a mirror which reflects the light transmitted through the specimen back into itself exactly. In this way, there is twofold transmission through the specimen with optimum illumination of the solid angle. In a laser scanning microscope, there are likewise two objectives with identical optical characteristics and at least one of the objectives is followed by a phase-conjugating or adaptive mirror.

[0001] This is a continuation application of parent U.S. patentapplication Ser. No. 09/658,321 filed Sep. 8, 2000, which claimspriority of DE 199 42 998.7 filed Sep. 9, 1999, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention is directed to a microscope in which a specimen isarranged between two objectives and can be observed with reflected lightas well as with transmitted light.

[0004] 2. Description of the Related Art

[0005] An important concern in further developments in microscopy atpresent is to provide and perfect methods and arrangements which make itpossible to observe objects by twofold transmission with reflected lightas well as with transmitted light, which serves to increase bothresolving capacity and contrast.

[0006] In this regard, there are already known arrangements in whichincident light transmitted by the object is reflected back to the rearof the object again by a reflecting device. The invention described inthe following also belongs to this class of arrangement.

[0007] An early solution using a reflecting device for the lighttransmitted by the object is described in DE 10 83 065. In this case,there is provided in the beam path behind the object a multiple cornerreflector or triple mirror which depolarizes the polarized light of thevertical or incident illumination and cooperates with a crossed analyzerarranged in the observation beam path in such a way that only thedepolarized beam component proceeding from the triple mirror can passthe analyzer and thus result in a transmitted-light image of the objectilluminated by incident light.

[0008] However, because of the influence of image errors (aberrations,etc.) and alignment or adjustment inaccuracies, the image of the objectto be observed has relatively weak lighting and poor contrast.

[0009] A further development in this respect according to DE 32 04 686A1 provides an optical system for transmitted-light microscopy withvertical or incident illumination in which it is attempted by means of aspecially constructed reflecting device to allow light beams which passthrough the object and are then reflected upon the object again to passidentical object points in both directions. For this purpose, it issuggested that, for example, an autocollimation system with optics whichimage the back of the object onto a plane mirror and image the occurringimage on the underside of the object is used as a reflecting device. Animproved contrast enhancement can be achieved with this system and thearrangement developed from it. In order to prevent aperture losses, theautocollimation system comprises, for example, two objectives withinfinite output back focal distance or output intersection length,wherein the object plane and the surface of the plane mirror lie in thefocal point of the two objectives.

[0010] However, the reflection carried out in this manner also hasalignment inaccuracies and image errors, as a result of which the lightis not exactly parallel after the second objective or the object to beobserved is not imaged onto itself with lateral and vertical precision.

OBJECT AND SUMMARY OF THE INVENTION

[0011] On this basis, it is the primary object of the invention toincrease efficiency in reflection and to ensure that the transmittedincident light is reflected back in itself again with high accuracy bythe reflecting device.

[0012] According to the invention, this object is met in a microscope inwhich a specimen is positioned between two objectives having opticalcharacteristics that are as identical as possible and at least one ofthe two objectives is followed by a mirror which reflects the lighttransmitted through the specimen back into itself exactly, so that thereis optimum illumination when light is transmitted twice through thepreparation. The image of the entire specimen volume obtained in thisway can be observed in the observation beam path of a microscope withfield-transmitting operation, wherein one of the two objectives servesas a microscope objective and the second objective is part of areflecting device.

[0013] The reflector surface of the mirror which is arranged subsequentto the reflecting objective is not plane as is the case in the priorart, but has a spherical curvature which, to a first approximation, isadapted to the wavefront of the reflecting objective. The reflectorsurface is preferably curved aspherically and is accordingly adapted tothe output wavefront of the reflecting objective.

[0014] In a particularly preferred embodiment of the invention, the twoobjectives have the same numerical aperture (NA) and also conform to oneanother as far as possible with respect to other characteristics,wherein both objectives are preferably constructed as planaprochromatswith a NA greater than or equal to 1.4.

[0015] In another possible embodiment of the invention, there is acoherent illumination source and the mirror provided in the reflectingdevice is constructed as a phase-conjugating mirror. Random disturbancesare optimized in real time with the phase conjugation in that anelectromagnetic wave is generated at the phase-conjugating mirrorsurface, which electromagnetic wave not only propagates in the oppositedirection, as is desired, but, beyond this, also has a reversed phasedistribution or an opposite sign of the phase.

[0016] Accordingly, in contrast to the conventional mirror, thedistortion of the wavefront is corrected, as a result of which the lightis imaged through the second objective again exactly in the focus of themicroscope objective. Compensation of losses which still occur in theprior art due to imaging errors and alignment inaccuracies issubstantially improved in this way.

[0017] When a laser source is used for illumination in connection withthe construction according to the invention, nonlinear phenomena can beutilized very favorably because the probability of multiphotonabsorption is substantially increased due to the bundling of the laserlight when passing through the specimen two times. When the laser lightis coupled into the microscope beam path via a dichroic beam splitter,the doubled wavelength which is diffusely reflected by the specimen canbe observed in a simple manner.

[0018] It also lies within the scope of the invention to provide anothermirror and to position this other mirror between the microscopeobjective and eyepiece in such a way that the specimen is imaged on thismirror through the microscope objective. This constructional variant isespecially relevant for fluorescence microscopy, wherein this mirrorpasses the illumination beam but does not pass a selected beam componentcoming from the specimen, e.g., the fluorescence radiation.

[0019] With an arrangement of this type, the two objectives which arelocated opposite one another symmetrically with respect to the specimenwith homogeneous immersion advantageously form an optical resonator bywhich very small phase interferences introduced in the resonator by thespecimen can be detected and can accordingly provide information aboutthe specimen with high resolution.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The invention will be described more fully in the following withreference to two embodiment examples.

[0021] In the Drawings:

[0022]FIG. 1 shows the arrangement according to the invention in afield-transmitting microscope;

[0023]FIG. 2 shows the arrangement according to the invention in aconfocal laser scanning microscope.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] In FIG. 1, a specimen 1 is received between the microscopeobjective 2 and another objective 3 which is identical to the microscopeobjective 2 with respect to its optical characteristics and which ispart of a reflecting device 4. Optimum resolutions result when, forexample, planaprochromats with a numerical aperture greater than orequal to 1.4 are used for both objectives 2, 3.

[0025] It is further advantageous when the preparation is receivedbetween two identical, high-grade cover glasses which ensure a perfectlysymmetrical beam path.

[0026] A mirror 5 which reflects the light transmitted through thespecimen 1 back into itself exactly is arranged in the reflecting device4 following the objective 3. The reflecting surface of the mirror 5 isnot plane, but rather has a sphere which is adapted to the wavefront ofthe objective 3 to a first approximation. In a particularly preferredmanner, the mirror surface is curved aspherically and adapted to theoutput wavefront of the objective 3.

[0027] Particularly in fluorescence microscopy, to which the presentexample pertains, the illumination light proceeding from a light source6 is reflected through an excitation filter 7 into the dichroic beamsplitter 8 and impinges on the specimen 1. The fluorescent light whichnow proceeds from the specimen 1 radiates in the entire solid angle andis accordingly detected by the microscope objective 2 as well as byobjective 3. After traversing the objective 3, the fluorescent light isparallel and impinges on the mirror 5 by which it is reflected backprecisely in the focus of the microscope objective 2 and is collected bythe microscope objective 2; after passing through the dichroic beamsplitter 8, the blocking filter 10 and the eyepiece 11, it is nowavailable for observation (or other evaluation).

[0028] Insofar as a laser is provided as illumination source and theobservation of the specimen 1 is carried out in coherent light, aphase-conjugating mirror can advantageously be provided as mirror 5, theuse of which ensures that the light impinging on the mirror surface isreflected back into itself in a highly accurate manner as intended.

[0029] The microscope can accordingly be operated with excitation bytransmitted light as well as reflected light. The excitation filter 7ensures that only the excitation beam reaches the microscope beam path 9from the illumination source 6. On the other hand, the blocking filter10 passes only the fluorescent light which is emitted by the specimenand which is to be evaluated.

[0030] The dichroic beam splitter 8 reflects the short-wave excitationlight coming from the illumination source 6 and passes the longer-wavefluorescent light proceeding from the specimen 1. The excitation lightis accordingly directed onto the specimen 1, while the fluorescentradiation collected by the microscope objective 2 and objective 3 passesthrough the beam splitter 8 and the blocking filter 10 to the eyepiece11 and into the eye of the observer.

[0031] As is indicated in FIG. 1, a partially-transmitting mirror 12 canbe provided in the microscope beam path 9 between the microscopeobjective 2 and the beam splitter 8. When this mirror 12 is constructedin such a way that it transmits the illumination wavelength but reflectsthe fluorescence wavelength back onto the specimen again, the microscopeobjective 2 and the objective 3 form the optical resonator, mentionedabove, by which very small phase interferences can be detected.

[0032] It is further shown in FIG. 1 that the reflecting device 4 can beexchanged with a photomultiplier 13. This can be accomplished by meansof a swiveling device so that the arrangement can be configured forphotometric transmitted-light measurements without cumbersomeconversion.

[0033]FIG. 2 shows a schematic view of a laser scanning microscope witha laser 14, a pinhole diaphragm 15 arranged in the laser beam path, ameasurement diaphragm 16 conjugated to the pinhole diaphragm 15, adetector 17, and a beam splitter 18.

[0034] The pinhole diaphragm 15 which is irradiated with laser light isimaged in the specimen 19, wherein the latter is illuminated with theintensity distribution of an Airy disk. In doing so, a point on thespecimen 19 is aimed for and an image of this point is formed on themeasurement diaphragm 16, wherein the position and size of this imagecan be evaluated by the detector 17. The measurement diaphragm 16 canonly pass light from an adjusted focal plane.

[0035] In this case, also, the specimen 19 is located between twoobjectives in a manner analogous to the first embodiment example(according to FIG. 1); one of these objectives forms the microscopeobjective 20 and another objective 21 is part of a reflecting device 22.A mirror 23 is arranged inside the reflecting device 22 followingobjective 21 and can be constructed as a phase-conjugating or adaptivemirror. With a mirror of this kind (as was already shown with referenceto the field-transmitting system), the laser light transmitted from thespecimen 19 is reflected back into itself exactly with respect todirection and phase front.

[0036] For the special case in which the mirror 23 is constructed as anadaptive mirror and is outfitted with actuating elements for deformationof its mirror surface, a control circuit 24 can be provided, as isindicated in FIG. 2, which is connected with the detector 17 on theinput side and with the actuating elements of the adaptive mirror 23 onthe output side.

[0037] For example, when the control circuit 24 is programmed in such away that it sends actuating signals to the adaptive mirror 23 dependingon the radiation intensity received by the detector 17, it is achievedin an advantageous manner that by appropriate actuation of the actuatingelements the curvature of the mirror surface is automatically adjustedsuch that the detector 17 can receive a fluorescent radiation of maximumintensity proceeding from the specimen 19.

[0038] As in the first embodiment example according to FIG. 1, theobjectives 20 and 21 located opposite one another symmetrically withrespect to the specimen 19 should also be identically constructed withrespect to their optical parameters and the specimen 19 should beprepared between two optically identical, high-grade cover glasses.

[0039] The adaptive mirror 23 can be constructed in the manner describedin detail in DE 26 31 551, for example, so that a more exhaustivetreatment herein can be dispensed with.

[0040] While the foregoing description and drawings represent thepresent invention, it will be obvious to those skilled in the art thatvarious changes may be made therein without departing from the truespirit and cope of the present invention.

[0041] It is possible, and also lies within the scope of this invention,to arrange diaphragms, Wollaston prisms, polarizers or analyzers and/orother subassemblies for optical contrasting in the beam path in a knownmanner. Any optical contrasting methods by which artificial contrastingcan be achieved without harmful intervention in the preparation can beused, i.e., darkfield methods, phase contrast methods in which phaseshifts are converted to brightness values, polarization contrast methodsfor observing birefringent specimens, generation of a differentialinterference contrast (DIC) and, above all, fluorescence contrasting.

[0042] This last embodiment can therefore be applied advantageouslyabove all in fluorescence microscopy because the fluorescent lightemitted by the specimen has a very low intensity in comparison to theexciting light. In the suggested manner, the fluorescent light that isnot directly detected by the microscope objective can be detected bymeans of the second objective in the reflecting device and is reflectedback again into the focus of the microscope objective. It is collectedin the latter and used as an additional basis for detection.

[0043] The invention is further directed to a laser scanning microscopein which a light-transmitting specimen is again positioned between twoobjectives with at least approximately identical optical characteristicsand a mirror is arranged following at least one objective, wherein thismirror is constructed as a phase-conjugating or adaptive mirror by whichthe wavefront of the reflected light is made to coincide with thewavefront of the transmitted light and the light is reflected back intoitself exactly with respect to direction and phase front.

[0044] In this way, the advantages of the arrangement according to theinvention can also be utilized particularly for confocal laser scanningmicroscopy. Optical scanning in which a light point deflected byoscillating mirrors or rotating polygon prism mirrors sweeps over theobject has proven successful in this connection. Pinhole diaphragmsconjugated in the illumination and observation beam path ensure thatonly the light from the respective adjusted focal plane reaches thedetector. In this way, spatially resolved and time-resolved data can beobtained in a known manner, but, thanks to the construction of thearrangement according to the invention, with substantially higherefficiency than in the known prior art.

[0045] As was already mentioned, the mirror surface of thephase-conjugating mirror is constructed in such a way that the wavefrontof a plane wave is changed after being reflected on the mirror surfacesuch that distortions are corrected and the reflected light is reflectedback into itself exactly.

[0046] On the other hand, the adaptive mirror which can be usedalternatively is provided with a deformable mirror surface arranged on adiaphragm, wherein a plurality of individual electrodes are locatedopposite the diaphragm on its side remote of the mirror surface andelectric voltage is applied to the diaphragm on the one hand and to theelectrodes on the other hand; the desired deformation of the diaphragmis triggered by changing the voltages, and accordingly the electrostaticforces, acting between the diaphragm and electrodes.

[0047] In this regard, control is carried out depending on the imagequality that has been achieved and, as the result of correspondingdeformation of the mirror surfaces, causes the light reflected by themirror to be reflected back exactly in itself and image errors andalignment inaccuracies are compensated.

[0048] The adaptive mirror can also be constructed in such a way thatthe diaphragm is connected, on its side remote of the mirror surface, toa plurality of individual piezoelectric drives and the deformation ofthe diaphragm is brought about by controlling the piezoelectric drivesin different ways.

[0049] The electrodes and/or the piezoelectric drives with which thedeformable mirror surfaces are coupled can communicate with a detectiondevice via an evaluating unit for a beam component which is coupled outof the observation beam path. The beam component is assessed accordingto intensity, for example, wherein an intensity signal is obtained andtaken as basis for determining an actuating signal for deformation ofthe mirror diaphragm.

[0050] This further development of the inventive idea is applicable influorescence microscopy in a particularly preferred manner in that theintensity of the fluorescent radiation proceeding from the specimen isassessed.

[0051] In other constructional variants of the invention relating tofield-transmitting and scanning systems, the reflecting device can beconstructed as a brightfield arrangement having two objectives whichtogether form an optical system with an infinite output intersectionlength.

[0052] Further, it is advantageous, particularly with respect toapplications for microphotometry, when the reflecting device can beswiveled out of the microscope beam path and a photomultiplier can beswiveled in in its place for transmitted-light detection. In this way,no cumbersome modification or adjustments are required for changing tophotometric measurements.

[0053] Another construction of the field-transmitting and laser scanningmicroscope consists in that at least one of the objectives is connectedwith an adjusting device for displacement in axial and/or radialdirection and the adjustment is carried out depending on the achievedimage quality or intensity and/or contrast. This adjusting possibilityis advantageous particularly for adjusting the optical resonatormentioned above. In this case, piezomechanical drive elements above allhave proven successful as actuating drives.

[0054] However, this possibility of axial and/or radial adjustmentserves not only for the adjustment of the optical resonator, but alsoopens the door to more or less novel contrasting methods, especiallywhen adjustment accuracies in the submicrometer range, preferably in therange of several hundred nm, are realized. Such accuracy can readily beachieved with piezo actuating elements, and phase interference anddifferential interference contrasting methods can be further developedin this way in terms of their efficiency.

[0055] Reference Numbers

[0056]1 specimen

[0057]2 microscope objective

[0058]3 objective

[0059]4 reflecting device

[0060]5 mirror

[0061]6 illumination

[0062]7 excitation filter

[0063]8 dichroic beam splitter

[0064]9 microscope beam path

[0065]10 blocking filter

[0066]11 eyepiece

[0067]12 partially transmitting mirror

[0068]13 photomultiplier

[0069]14 laser

[0070]15 pinhole diaphragm

[0071]16 measurement diaphragm

[0072]17 detector

[0073]18 beam splitter

[0074]19 specimen

[0075]20 microscope objective

[0076]21 objective

[0077]22 reflecting device

[0078]23 mirror

[0079]24 control circuit

What is claimed is:
 1. A microscope comprising: two objectives betweenwhich a light-transmitting specimen is arranged; said objectives havingat least substantially identical optical characteristics; and at leastone of said two objectives being followed by a mirror for reflectinglight transmitted through the specimen back into itself exactly.
 2. Themicroscope according to claim 1, wherein the two objectives have thesame numerical aperture and the same other characteristics, wherein bothobjectives are constructed as planapochromats with a numerical aperturegreater than or equal to 1.4.
 3. The microscope according to claim 1,with incident illumination and field transmission of an imageinformation, wherein one of the objectives serves as a microscopeobjective and the second objective is part of a reflecting devicethrough which the specimen is imaged onto itself with lateral andvertical accuracy.
 4. The microscope according to claim 1, whereindiaphragms, Wollaston prisms, polarizers or subassemblies for opticalcontrasting are arranged in a beam path.
 5. The microscope according toclaim 1, but with a coherent illumination source in which one of themirrors is constructed as a phase-conjugating mirror.
 6. The microscopeaccording to claim 1, wherein a dichroic beam splitter is provided forreflecting into the illumination source.
 7. The microscope according toclaim 1, wherein another mirror is provided between the microscopeobjective and eyepiece, the specimen being imaged on this mirror throughthe microscope objective, wherein this mirror passes the illuminationbeam but does not pass a selected beam component, preferably fluorescentradiation, coming from the specimen.
 8. The microscope according toclaim 1, constructed as a laser scanning microscope, wherein one of theobjectives serves as a microscope objective and the second objective ispart of a reflecting device having a phase-conjugating mirror or anadaptive mirror by which the wavefront of the reflected light is made tocoincide with the wavefront of the transmitted light.
 9. The microscopeaccording to claim 8, wherein the adaptive mirror (23) is provided witha deformable mirror surface arranged on a diaphragm, and a plurality ofindividual electrodes are located opposite the diaphragm on its sideremote of the mirror surface, and electric voltage is applied to thediaphragm on the one hand and to the electrodes on the other hand, andthe deformation of the diaphragm is brought about by changing thevoltages and electrostatic forces acting between the diaphragm andelectrodes.
 10. The microscope according to claim 9, wherein theelectrodes communicate with a detection device for a beam componentwhich is coupled out of an observation beam path, with fluorescentradiation proceeding from the specimen.
 11. The microscope according toclaim 1, wherein the reflecting device is constructed as a brightfieldarrangement having two objectives which together form an optical systemwith an infinite output intersection length.
 12. The microscopeaccording to claim 1, wherein the reflecting device can be swiveled outof the microscope beam path and a photomultiplier can be swiveled in itsplace for transmitted-light detection.
 13. The microscope according toclaim 1, wherein at least one of the objectives is connected withadjusting devices for displacement in axial and/or radial direction andthe adjustment is carried out depending on the observation beam pathwith respect to its intensity or contrast.
 14. The microscope accordingto claim 12, wherein the adjusting devices are coupled with driveelements.
 15. The microscope according to claim 12, wherein said driveelements are piezomechanical drive elements.
 16. The microscopeaccording to claim 1, wherein there is a detector for a beam componentwhich is coupled out of an observation beam path, with fluorescentradiation proceeding from the specimen.
 17. The microscope according toclaim 8, wherein the adaptive mirror is provided with a deformablemirror surface arranged on a diaphragm, the diaphragm is connected, onits side remote of the mirror surface, to a plurality of individualpiezoelectric drives and the deformation of the diaphragm is broughtabout by controlling the piezoelectric drives.
 18. The microscopeaccording to claim 17, wherein the piezoelectric drives communicate witha detection device for a beam component which is coupled out of theobservation beam path, with fluorescent radiation proceeding from thespecimen.
 19. A microscope comprising: two objectives between which alight-transmitting specimen is arranged; said objectives having at leastsubstantially identical optical characteristics; and at least one ofsaid two objectives being followed by a phase-conjugating mirror forreflecting light transmitted through the specimen back into itselfexactly with respect to direction and phase front; and a detector forreceiving reflected specimen fluorescent radiation from the lighttransmitting specimen.
 20. A confocal laser scanning microscope forexamining a light transmitting specimen comprising: a laser forproviding excitation light to the light transmitting specimen to inducefluorescence in the specimen whereupon the excitation light and thefluorescence is transmitted through the specimen; two objectives betweenwhich the light-transmitting specimen is arranged; a first pinholediaphragm located between the laser and the objectives; said objectiveshaving at least substantially identical optical characteristics; atleast one of said two objectives being followed by an optically adaptivemirror or phase conjugating mirror for reflecting the excitation lightand the fluorescence transmitted through the specimen back into thespecimen exactly to improve contrast; a detector for receiving specimenfluorescent radiation from the light transmitting specimen; a secondpinhole diaphragm located between the objectives and the detector.